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Cfr is a radical SAM (S-adenosyl-L-methionine) methyltransferase capable of conferring resistance to phenicols, linocosmides, oxazolidinones, pleuromutilins and streptogramin A (termed the PhLOPSA phenotype) via the C8 methylation of 23s rRNA residue A2503. Radical SAM enzymes utilise a [4Fe-4S] cluster to coordinate the reductive cleavage of SAM producing a 5’Deoxyadenosyl radical, which is used to catalyse reactions at inherently unreactive carbon centres. To date, attempts to crystallise Cfr have been unsuccessful and all structural data originate from RlmN, a Cfr homolog with no resistance phenotype capable of methylating position C2 of the same residue. To address the void of structural knowledge regarding Cfr, four Cfr-like enzymes were selected for investigation into their biochemical characteristics and potential for crystallisation: the Cfr(B) and Cfr(C) alleles originally identified in resistant pathogens, and the PbCfr (Paenibacillus spp.) and SbCfr (Sphaerobacter thermophilus) homologues from thermophilic organisms. Work presented here shows expression of SbCfr and Cfr in recombinant E. coli BL21* and anaerobic purification by immobilised metal ion chromatography, yielding 10.25 and 22.4 mg.L-1 E. coli culture, respectively. UV-vis spectra of SbCfr display a 420nm peak consistent with the presence of intact [4Fe-4S] clusters before and after reconstitution, while anaerobic activity assays show s-adenosylhomocysteine (SAH) and 5’ deoxyadenosine (DOA) formation by both reconstituted and unreconstituted SbCfr. Activity assays and circular dichroism spectroscopy indicate SbCfr activity up to 55ºC and stability up to 65ºC. However, SbCfr did not confer the PhLOPSA phenotype on recombinant E. coli, while efforts to use site-directed mutants to trap covalent RNA complexes, indicative of RlmN-like activity, were similarly unsuccessful. Although crystallisation trials of Cfr were not successful, SbCfr was successfully crystallised and the struct

Cfr is a radical SAM (S-adenosyl-L-methionine) methyltransferase capable of conferring resistance to phenicols, linocosmides, oxazolidinones, pleuromutilins and streptogramin A (termed the PhLOPSA phenotype) via the C8 methylation of 23s rRNA residue A2503. Radical SAM enzymes utilise a [4Fe-4S] cluster to coordinate the reductive cleavage of SAM producing a 5’Deoxyadenosyl radical, which is used to catalyse reactions at inherently unreactive carbon centres. To date, attempts to crystallise Cfr have been unsuccessful and all structural data originate from RlmN, a Cfr homolog with no resistance phenotype capable of methylating position C2 of the same residue. To address the void of structural knowledge regarding Cfr, four Cfr-like enzymes were selected for investigation into their biochemical characteristics and potential for crystallisation: the Cfr(B) and Cfr(C) alleles originally identified in resistant pathogens, and the PbCfr (Paenibacillus spp.) and SbCfr (Sphaerobacter thermophilus) homologues from thermophilic organisms. Work presented here shows expression of SbCfr and Cfr in recombinant E. coli BL21* and anaerobic purification by immobilised metal ion chromatography, yielding 10.25 and 22.4 mg.L-1 E. coli culture, respectively. UV-vis spectra of SbCfr display a 420nm peak consistent with the presence of intact [4Fe-4S] clusters before and after reconstitution, while anaerobic activity assays show s-adenosylhomocysteine (SAH) and 5’ deoxyadenosine (DOA) formation by both reconstituted and unreconstituted SbCfr. Activity assays and circular dichroism spectroscopy indicate SbCfr activity up to 55ºC and stability up to 65ºC. However, SbCfr did not confer the PhLOPSA phenotype on recombinant E. coli, while efforts to use site-directed mutants to trap covalent RNA complexes, indicative of RlmN-like activity, were similarly unsuccessful. Although crystallisation trials of Cfr were not successful, SbCfr was successfully crystallised and the struct

Throughout the nineteenth century, the prayers, addresses, memorials, legal briefs, testimony and delegations of the Turtle Mountain Chippewa exemplified Edward Said's insight that "nations themselves are narrations." Their legal struggle for land and sovereignty derived from "the power to narrate" their own side of the story. This tribal case study confirms that the Turtle Mountain Chippewa are a powerful people with a compelling history. An adherence to the Native viewpoint is required to re-examine the formulation and implementation of nineteenth century federal Indian policy. This more inclusive approach can help everyone gain a broader perspective on the history of European American/American Indian relations.

In a lecture delivered at Queen's University in Belfast in 1955, historian Michael Roberts argued that in the one-hundred years between 1560 and 1660, a number of critical changes and innovations had occurred in tactics, strategy, army size, and sociopolitical institutions, which taken together amounted to a "military revolution" in Early Modern Europe. Roberts held that Gustavus Adolphus's combination of linear formations and improved firepower had revolutionized tactics, which in turn created fresh strategic possibilities. The broader range of strategic options then led to the need for large standing armies, which ultimately forced states to develop political and social institutions to oversee and supply them. Together, these changes wrought a revolution in the Early Modern style of waging war. With its publication three years later, Roberts' lecture captured the interest of the historical community and excited further research into the nature and effect of military change in the 16th and 17th centuries. Over the years a number of historians of Europe's Early Modern period, notably Geoffrey Parker and J. R. Hale, have modified and added to Roberts' original thesis. Despite these changes, however, Roberts' basic argument has stood the test of time. In a similar manner, contemporary soldiers and scholars are struggling to appreciate the significance of the Persian Gulf War in terms of its critical tactical, strategic, and sociopolitical aspects and its impact on future wars. Simply put, the question has become whether the Gulf War represents a new style or form of warfare and, if so, what effect this new style of warfare is likely to have on future warfighting. At the heart of this question lies, once again, the issue of military change., Published in Parameters, v22 p70-79, Winter 1992-1993.